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SUMMARYNucleated cells employ several strategies to evade killing by homologous complement. We studied complement resistance in the human carcinoma cell lines (CA) T47D (mammary), SKOV3 (ovarian), and PC-3 (prostate) with emphasis on the following mechanisms of defense: 1. Expression and shedding of the membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59; 2. Resistance based on protein phosphorylation; 3. Cell surface expression of sialic acid residues; 4. Desensitization to complement upon exposure to sublytic complement doses. Anti-mCRP antibody blocking experiments demonstrated that CD59 is the main mCRP protecting these CA from complement. Soluble CD59 was also found in supernates of PC-3 > SKOV3 > T47D cells. Second, inhibitors of PKC, PKA and MEK sensitized the CA to lysis, thus implicating these protein kinases in CA complement resistance. Third, removal of sialic acid residues with neuraminidase also sensitized CA to lysis. Finally, exposure of CA to sublytic doses of complement conferred on them enhanced resistance to lytic complement doses in a PKC-dependent process. Combined treatment of CA with anti-CD59 antibodies, PD98059 (a MEK inhibitor) and neuraminidase produced a large enhancement in CA sensitivity to complement. Our results show that CD59 and sialic acid residues present on the cell surface, and intracellular processes involving protein phosphorylation act additively to secure CA resistance to complement-mediated lysis. Therefore, the effectiveness of antibody-and complement-based cancer immunotherapy will markedly improve by suppression of the various complement resistance mechanisms.

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Resistance of tumor cells to lysis by complement is generally attributed to several protective mechanisms. The relative impact of these mechanisms in the same tumor cell, however, has not been assessed yet. We have analyzed the interaction of the human erythroleukemia tumor cell line K562 with human complement. K562 cells express the membrane complement regulatory proteins CD59, CD55 and CD46. As shown here for the first time, K562 also spontaneously release the soluble regulators C1 inhibitor, factor H, and soluble CD59. Complement resistance of K562 cells is augmented upon treatment with PMA, TNF or even with sublytic complement. Unlike TNF and sublytic complement, PMA enhanced the expression of membrane-bound CD55 and CD59 and led to increased secretion of soluble CD59. In addition, we show that complement-resistant K562 cells express a membrane-associated proteolytic activity, higher than the complement-sensitive K562/S cells. Treatment of complement-resistant K562 cells with serine protease inhibitors enhance their sensitivity to complement-mediated lysis. Inhibitors of protein kinase C (PKC) also sensitize K562 cells to complement lysis, implicating PKC-mediated signaling in cell resistance to complement. Neutralization of the CD55 and CD59 but not of CD46 regulatory activity with specific antibodies significantly increases complement-mediated K562 cell lysis. Treatment of K562 cells with a mixture of inhibitory reagents results in a significant additive enhancing effect on complement-mediated lysis of K562. In conclusion, K562 cells resist a complement attack by concomitantly using multiple molecular evasion strategies. Future attempts in antibody-based tumor therapy should include strategies to interfere with those resistance mechanisms.

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